AU5774101A - Method for producing threonine and isoleucine - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): AJINOMOTO CO., INC.

Invention Title: METHOD FOR PRODUCING THREONINE AND ISOLEUCINE The following statement is a full description of this invention, including the best method of performing it known to me/us: Method for Producing Threonine and Isoleucine Background of the Invention Field of the Invention The present invention relates to a technique used in fermentation industry, and it relates to a bacterium belonging to the genus Escherichia that produces Lthreonine or L-isoleucine and a method for producing Lthreonine or L-isoleucine using the bacterium.

Description of the Related Art Industrial production of L-amino acids such as Lthreonine and L-isoleucine has conventionally been attained by fermentation method using microorganisms such as coryneform bacteria and bacteria belonging to the genus Escherichia having ability to produce such Lamino acids. As these amino acid producing bacteria, there are used strains isolated from nature, artificial 20 mutant strains thereof or recombinant strains thereof in which L-amino acid biosynthesis enzymes are enhanced by genetic recombination in order to obtain improved productivity.

Specifically, as methods for producing L-threonine, 25 there have been disclosed a method utilizing a mutant strain of bacterium belonging to the genus Escherichia in Japanese Patent Laid-open Publication (Kokai) No. 304969, methods utilizing recombinant Escherichia coli strains in Japanese Patent Publication Nos. 1-29559, 2- 109985, 56-15696 and International Patent Publication in Japanese (Kohyo) No. 3-501682, and a method utilizing a mutant strain of Corynebacterium bacterium in Japanese Patent Laid-open Publication No. 62-239996, and a method utilizing a mutant strain of Corynebacterium bacterium is reported in Japanese Patent Laid-open Publication No.

61-195695. Further, methods for producing L-threonine by utilizing strains transformed with recombinant plasmids containing the threonine operon have been disclosed in Japanese Patent Laid-open Publication Nos.

55-131397, 59-31691, 56-15696 and International Patent Publication in Japanese No. 3-501682.

Further, as methods for producing L-isoleucine, •there have been disclosed a method utilizing Escherichia coli in Japanese Patent Laid-open Publication No. 130882, a method utilizing a recombinant strain of Escherichia coli in Japanese Patent Laid-open 20 Publication No. 2-458, a method utilizing mutant strain of Corynebacterium bacterium in Japanese Patent Publication No. 3-62395, and a method utilizing a recombinant strain of Corynebacterium bacterium in Japanese Patent Publication (Kokoku) No. 5-47196. It is 25 also known that L-isoleucine producing ability can be imparted by introducing thrABC operon containing thrA gene coding for aspartokinase I-homoserine dehydrogenase I derived from Escherichia coli, of which inhibition by L-threonine is substantially desensitized, and ilvGMEDA operon containing ilvA gene coding for threonine deaminase, of which inhibition by L-isoleucine is substantially desensitized, and from which a region required for attenuation is removed (see Japanese Patent Laid-open Publication No. 8-47397) Meanwhile, the sequence of phosphoenolpyruvate carboxylase gene of Escherichia coli is known (Fujita, Miwa, Ishijima, Izui, K. and Katsuki H. J.

Biochem. 95, 909-916 (1984)), and there have been disclosed phosphoenolpyruvate carboxylase of which feedback inhibition by aspartic acid is substantially desensitized and a method for utilizing a gene therefor (W095/06114). Further, there is also known an example of enhancement of phosphoenolpyruvate carboxylase gene with the purpose of enhancement of L-glutamic acid producing ability of coryneform bacteria (Japanese Patent Laid-open Publication No. 60-87788). Furthermore, 20 there have also been disclosed techniques of improving amino acid producing ability by enhancing a phosphoenolpyruvate carboxylase gene together with other enzyme genes. For example, an example has been reported, in which L-glutamic acid producing ability was enhanced 25 by enhancing glutamate dehydrogenase gene, citrate synthetase gene and phosphoenolpyruvate carboxylase gene in coryneform bacteria in which a-ketoglutarate 4 dehydrogenase gene was deleted (W096/06180). As for Escherichia coli, it has been disclosed that L-threonine producing ability was not significantly increased even if a wild-type phosphoenolpyruvate carboxylase gene was introduced into an L-threonine producing strain of Escherichia coli, B-3996, which was transformed with a recombinant plasmid containing the threonine operon (W095/06114).

Further, it has also been disclosed that ability to produce substances such as amino acids can be improved by increasing enzymatic activity of nicotinamide nucleotide transhydrogenase (also referred to as "transhydrogenase" hereafter) in microbial cells, and increasing reduced type nicotinamide adenine dinucleotide phosphate producing ability (W095/11985).

In this reference, it is also mentioned an example of improvement of L-threonine producing ability by enhancing a transhydrogenase gene in Escherichia coli transformed with a recombinant plasmid containing the S 20 threonine operon. As an amino acid of which productivity is improved by elevation of transhydrogenase activity, L-isoleucine was mentioned.

Summary of the Invention An object of the present invention is to improve ability to produce L-threonine or L-isoleucine of bacteria belonging to the genus Escherichia.

The inventors of the present invention found that the ability to produce L-threonine or L-isoleucine was markedly increased by enhancing both of phosphoenolpyruvate carboxylase activity and transhydrogenase activity, and further found that the producing ability was further improved by enhancing aspartase activity. Thus, they accomplished the present invention.

That is, the present invention provides the followings.

A bacterium belonging to the.genus Escherichia, which has an ability to produce L-threonine or Lisoleucine, and' in which intracellular phosphoenolpyruvate carboxylase activity and transhydrogenase activity are enhanced.

The bacterium belonging to the genus Escherichia according to in which activity of an enzyme or enzymes encoded by threonine operon or a part thereof is 20 enhanced, and which has L-threonine producing ability.

The bacterium belonging to the genus Escherichia according to wherein the threonine operon consists of thrABC.

The bacterium belonging to the genus Escherichia 25 according to in which activity of an enzyme or enzymes encoded by ilv operon or a part thereof is enhanced, and which has L-isoleucine producing ability.

The bacterium belonging to the genus Escherichia according to any one of to wherein aspartase activity is enhanced.

The bacterium belonging to the genus Escherichia according to any one of to wherein activity of each enzyme is enhanced by increasing copy number of a gene or operon coding for each enzyme, or modifying an expression regulatory sequence so that intracellular expression of the gene or operon should be enhanced.

The bacterium belonging to the genus Escherichia according to wherein the gene is derived from a bacterium belonging to the genus Escherichia.

A method for producing L-threonine or L-isoleucine, which comprises'culturing a bacterium belonging to the genus Escherichia according to'any one of to in a medium to produce and accumulate L-threonine or Lisoleucine in the medium, and collecting the L-threonine or L-isoleucine from the medium.

According to the present invention, L-threonine or 20 L-isoleucine producing ability of bacteria belonging to the genus Escherichia can be improved.

Brief Explanation of the Drawings 25 Fig. 1 shows the construction of the plasmid pMW118::aspA containing aspA gene.

Fig. 2 shows the construction of the plasmid containing pntAB gene and ppc gene (pPTS).

Fig. 3 shows the construction of the plasmid containing aspA gene and ppc gene (pAPW).

Fig. 4 shows the construction of the plasmid containing aspA gene, pntAB gene and ppc gene (pAPT).

Fig. 5 shows the construction of the plasmid pHSGSK.

Fig. 6 shows the construction of the plasmid pdGMl.

Fig. 7 shows the construction of the plasmid pMWGMA2.

Fig. 8 shows the construction of the plasmid Fig. 9 shows the construction of pMWD5-ppc, pMWD5-PTS and Detailed Description of the Invention Hereafter, the present invention will be explained in detail.

A bacterium belonging to the genus Escherichia of the present invention is a bacterium belonging to the genus Escherichia which has an ability to produce Lthreonine or L-isoleucine, and has enhanced intracellular phosphoenolpyruvate carboxylase (also abbreviated as "PEPC" hereafter) activity and 25 transhydrogenase (also abbreviated as "THY" hereafter) activity.

As the bacteria belonging to the genus Escherichia, specifically, those mentioned in the work of Neidhardt et al. (Neidhardt, F.C. et al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington 1208, Table 1) can be used. For example, Escherichia coli can be mentioned.

The expression "having ability to produce Lthreonine or L-isoleucine" used herein means that, when the bacterium of interest is cultured in a medium, it shows an ability to accumulate L-threonine or Lisoleucine in the medium. This L-threonine or Lisoleucine producing ability may be a property possessed by a wild strain or a property imparted or enhanced by breeding.

In the bacterium belonging to the genus Escherichia of the present invention, intracellular aspartase (L-aspartate ammonia-lyase, also referred to as "AspA" hereinafter) activity may be further enhanced.

In order to enhance activity of PEPC, THY or AspA in bacteria belonging to the genus Escherichia, a gene S 20 coding for PEPC, THY or AspA can be cloned on a suitable plasmid, and a bacterium belonging to the genus Escherichia that serves as a host can be transformed with the obtained plasmid. This increases copy number of a gene coding for PEPC, THY or AspA (hereafter 25 abbreviated as "ppc gene", "pntAB gene" and "apsA gene", respectively, in that order) in the transformant, and as a result, the activity of PEPC, THY or AspA is enhanced.

The ppc gene, pntAB gene and apsA gene are introduced into a bacterium belonging to the genus Escherichia as a combination of the ppc gene and pntAB gene, or a combination of these genes and the aspA gene.

These genes may be introduced into a host as one kind of plasmid in which two or three of the genes are cloned, or two or three kinds of plasmids that can coexist, in which the genes are respectively cloned.

The enhancement of PEPC, THY or AspA activity can also be attained by allowing existence of multiple copies of the ppc gene, pntAB gene or apsA gene on chromosomal DNA of the original parent strain that serves as a host. In order to introduce multiple copies of the ppc gene', pntAB gene or apsA gene into chromosomal DNA of a bacterium'belonging to the genus Escherichia, a sequence of which multiple copies exist in the chromosomal DNA, for example, repetitive DNA, inverted repeats existing at the end of a transposable Selement etc., can be used. Alternatively, it is also 20 possible to incorporate the ppc gene, pntAB gene or apsA gene into transposon, and allow its transfer to introduce multiple copies of each gene into the chromosomal DNA. By either method, the number of copies of the ppc gene, pntAB gene or apsA gene within cells of 25 the transformant strain increases, and as a result, PEPC, THY or AspA activity is enhanced.

The enhancement of PEPC, THY or AspA activity can also be attained by, besides being based on the aforementioned gene amplification, replacing an expression regulatory sequence of ppc gene, pntAB gene or apsA gene such as a promoter with a stronger one (see Japanese Patent Laid-open Publication No. 1-215280).

For example, lac promoter, trp promoter, trc promoter, tac promoter, P, promoter and PL promoter of lambda phage, tet promoter, amyE promoter, spac promoter and so forth are known as strong promoters. Substitution of these promoters enhances expression of the ppc gene, pntAB gene or apsA gene, and hence the PEPC, THY or AspA activity is enhanced. Enhancement of an expression regulatory sequence may be combined with increasing copy number of the ppc gene, pntAB gene or apsA gene.

The organism as the source of the ppc gene, pntAB gene or apsA gene may be any organism having the PEPC, THY or AspA activity. Particularly preferred are bacteria that are prokaryotes, for example, bacteria belonging to the genus Enterobacter, Klebsiella, Erwinia, 20 Serratia, Escherichia, Corynebacterium, Brevibacterium or Bacillus. As a specific example, Escherichia coli can be mentioned. The ppc gene, pntAB gene or apsA gene can be obtained from chromosomal DNA of such microorganisms as mentioned above.

25 The ppc gene of Escherichia coli can be obtained from a plasmid having this gene, plasmid pS2 (Sabe, H.

et al., Gene, 31, 279 (1984)) or pT2. By digesting pS2 with AatII and AflII, a DNA fragment containing the ppc gene can be obtained. A DNA fragment having the ppc gene can also be obtained by digesting pT2 with SmaI and ScaI. The E. coli F15 strain (AJ12873) harboring pT2 was deposited on July 15, 1993 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology Higashi 1-chome, Tsukubashi, Ibaraki-ken, Japan, postal code: 305-8566) (currently, the independent administrative corporation, the National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibarakiken, Japan, postal code: 305-5466) and received an accession number of FERM P-13752. Then, it was transferred to an international deposit under the provisions of the Budapest treaty on July 11, 1994, and received an accession number of FERM BP-4732.

The pntAB gene can be obtained by digesting the plasmid pMW::THY (W095/11985) containing the gene with 20 SmaI and HindIII. The Escherichia coli AJ12929 strain harboring pMW::THY was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 25 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on October 4, 1993, and received an accession number of FERM P-13890. Then, it was transferred from the above 6' 1 12 original deposit to an international deposit under the provisions of the Budapest Treaty on September 14, 1994, and received an accession number of FERM BP-4798. The transhydrogenase of Escherichia coli consists of two subunits, which are encoded by pntA and pntB, respectively.

While the bacterium belonging to the genus Escherichia of the present invention is not particularly limited so long as it has the L-threonine or Lisoleucine producing ability, specific examples thereof include, for example, bacteria belonging to the genus Escherichia imparted.with the L-threonine producing ability by enhancing activity of an enzyme encoded by the threonine operon or a part thereof and in addition, bacteria belonging to the genus Escherichia imparted with the L-isoleucine producing ability by enhancing activity of an enzyme encoded by the ilv operon or a part thereof.

The threonine operon or a part thereof may be, for S 20 example, thrABC or a part thereof. The ilv operon or a part thereof may be, for example, ilvGMEDA or a part thereof.

As Escherichia coli having L-threonine producing ability, there can be specifically mentioned Escherichia 25 coli VKPM B-3996 (deposited on November 19, 1987 at All- Union Scientific Center of Antibiotics, Nagatinskaya Street 3-A, 113105, Moscow, Russian Federation with a registration number of RIA 1867, see U.S. Patent No.

5,175,107), Escherichia coli AJ11335 (Japanese Patent Laid-open Publication No. 55-131397) and so forth. The VKPM B-3996 strain harbors a plasmid (International Patent Publication W090/04636), which is obtained by inserting a threonine biosynthesis system gene (threonine operon: thrABC) into a wide host-range vector plasmid having a streptomycin resistance marker, pAYC32 (see Chistorerdov, Tsygankov, Plasmid, 1986, 16, 161-167). The feedback inhibition by Lthreonine of the aspartokinase I-homoserine dehydrogenase I encoded by thrA in that operon is desensitized.

As bacteria belonging to the genus Escherichia having L-isoleucine producing ability, the Escherichia coli KX141 (VKPM B-4781, see European Patent Laid-open Publication No. 519,113) and Escherichia coli AJ12919 (Japanese Patent Laid-open Publication No. 8-47397) can be mentioned. The VKPM B-3996 strain in which the ilv operon is amplified is also a preferred L-isoleucine producing bacterium.

The threonine operon contains the thrA, thrB and thrC genes, and they code for aspartokinase I-homoserine dehydrogenase I, homoserine kinase and threonine 25 synthase, respectively, in that order. As for these enzymes, it is preferred that the inhibition of aspartokinase I-homoserine dehydrogenase I by Lthreonine should be substantially desensitized.

The ilvGMEDA operon contains the ilvG, ilvM, ilvE, ilvD and ilvA genes, and they code for the large subunit, small subunit, transaminase, dihydroxy-acid dehydratase and threonine deaminase of isozyme II of acetohydroxyacid synthase, respectively, in that order. Since the ilvGMEDA operon is under control (attenuation) of expression of the operon by L-valine and/or L-isoleucine and/or L-leucine, a region required for the attenuation may be removed or mutated in an L-isoleucine producing bacterium in order to desensitize suppression of the expression by the produced L-isoleucine. As the ilvGMEDA operon, those derived from bacteria belonging to the genus Escherichia, in particular, the ilvGMEDA operon derived from E. coli, can be mentioned. The ilvGMEDA operon is detailed in W096/26289. As for the ilvGMEDA operon, it is preferred that the region required for attenuation should be removed, and among the enzymes encoded by this operon, inhibition of threonine deaminase by L-isoleucine should be a substantially desensitized (see Japanese Patent Laidopen Publication No. 8-47397).

Enhancement of activities of the enzymes encoded by the threonine operon or ilv operons or a part thereof 25 may be attained in the same manner as that for PEPC, THY and AspA.

In a microorganism used for the present invention,

I

if a gene for an enzyme responsible for a pathway involved in biosynthesis of target amino acid is enhanced, or a gene or operon coding for a desensitized type (inhibition desensitized type) enzyme of an enzyme suffering from feedback inhibition is introduced, the Lamino acid producing ability may further be improved.

Threonine or isoleucine can be produced by culturing a bacterium belonging to the genus Escherichia in which PEPC and THY as well as AspA, if required, are enhanced as described above and which has an ability to produce L-threonine or L-isoleucine in a medium to produce and accumulate threonine.or isoleucine in the medium, and collecting the threonine or isoleucine from the medium.

The medium used for the culture may be a usual medium containing a carbon source, nitrogen source, inorganic ions, and other organic components as required.

As the carbon source, it is possible to use sugars such as glucose, lactose, galactose, fructose and starch hydrolysate; alcohols such as glycerol and sorbitol; or organic acids such as fumaric acid, citric acid and succinic acid.

As the nitrogen source, it is possible to use inorganic ammonium salts such as ammonium sulfate,

*SSSSS

25 ammonium chloride or ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; or aqueous ammonia.

As for the organic trace nutrients, it is desirable to add required substances such as vitamin B,, yeast extract and so forth in a suitable amount. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and so forth are added.

Culture is preferably carried out under an aerobic condition for 16-72 hours. The culture temperature is controlled to be 25 0 C to 45 0 C, and pH is controlled to be 5 to 8 during the culture. Inorganic or organic, acidic or alkaline substances as well as ammonia gas and so forth can be used for pH adjustment.

Collection of L-threonine or L-isoleucine from fermented liquor is usually carried out by a combination of an ion exchange resin technique, precipitation and other known techniques.

0o o @600 Best Mode for Carrying out the Invention The present invention will be further specifically explained hereinafter with reference to the following examples.

00* Example 1: Production of plasmids containing various 25 enes Production of plasmid containing aspA gene (pMW118::aspA) 1 17 A DNA fragment containing the aspA gene was amplified by PCR using chromosomal DNA of the Escherichia coli W3110 strain as a template and the following primers.

Primer 1: 5'-TGATCAGCGAAACACTTTTA-3' (SEQ ID NO: 1) Primer 2: 5'-CAGCAAACTATGATGAGAA-3' (SEQ ID NO: 2) The obtained amplified fragment was inserted into the SmaI cleavage site of pMW118 (Nippon Gene) to obtain pMW118::aspA (Fig. 1).

Production of plasmid containing pntAB gene and ppc gene (pPTS) The plasmid pMW::THY containing the pntAB gene described in W095/11985 was digested with SmaI and HindIII, and a DNA fragment containing pntAB was collected. Then, the plasmid pppc containing the ppc gene described in W095/16042 was digested with XbaI.

After the both ends were blunt-ended, it was further digested with HindIII, and inserted with the above DNA fragment containing pntAB at the cleavage site to obtain

S

a plasmid pPTS (Fig. 2).

eae. Production of plasmid containing aspA gene and ppc gene (pAPW) 25 pMWI18::aspA was digested with SacI, and the both ends were blunt-ended. It was further digested with HindIII to obtain a DNA fragment containing aspA. Then, 18 the aforementioned pppc was digested with XbaI, and the both ends were blunt-ended. It was further digested with HindIII, and inserted with the aforementioned DNA fragment containing aspA at the cleavage site to obtain pAPW (Fig. 3).

Production of plasmid containing aspA gene, pntAB gene, and ppc gene (pAPT) A DNA fragment containing pntAB was obtained by digesting pMW::THY with SmaI and HindIII. Then, the aforementioned pAPW was digested with XbaI, and the both ends were blunt-ended. It was further digested with HindIII and inserted with the aforementioned pntAB at the cleavage site to obtain pAPT (Fig. 4).

Production of plasmid containing ilvGMEDA operon A DNA fragment containing ilvGMEDA operon was prepared from the plasmid pMWD5 containing the ilvGMED operon, which is disclosed in W096/26289. The plasmid pMWD5 was constructed as follows.

The chromosomal DNA was extracted from Escherichia S• coli MI162. The chromosomal DNA was cleaved with restriction enzyme HindIII. The length of a HindIII-HindIII DNA fragment including ilvGM genes was found to be 4.8 kb. Therefore, the HindIII-HindIII DNA fragment with approximately 4.8 kb and the DNA fragment 19 obtained by digestion of the plasmid vector pBR322 (purchased form Takara Shuzo, Co., Ltd.) with HindIII, were ligated.

The resulting DNA-ligated mixture was induced into Escherichia coli MI162 which is an acetohydroxy-acid synthase-deficient strain. The strains in which the deficiency of acetohydroxy-acid synthase was complemented by transformation were selected and the plasmid structure was isolated from the selected strains.

The results of the analysis of the plasmid revealed that a 4.8-kb DNA fragment containing the ilvGM gene and a portion of 5'-terminal of ilvE gene was inserted into the HindIII site of the pBR322. The plasmid was termed pBRGM7.

15 The synthetic oligonucleotides shown in SEQ ID NO:3 and NO:4 were synthesized with reference to the DNA sequence of the ilvGM gene described in Gene, 97, 21, (1991), Pro. Natl. Acad. Sci. 78, 922, (1981) and J. Bacteriol., 149, 294, (1982). DNA was amplified by the PCR method, using both oligonucleotides as primers and chromosomal DNA of MI162 strain as a template. The amplified fragment was termed Fragment Similarly, the synthetic oligonucleotides shown in SEQ ID NO:5 and NO:6 were synthesized with reference to the DNA sequence described in Gene, 97, 21, (1991), Pro.

Natl. Acad. Sci. 78, 922, (1981) and J.

Bacteriol., 149, 294, (1982). DNA was amplified by the PCR method, using both synthesized DNAs as primers and chromosomal DNA of the MI162 strain as a template. The amplified DNA fragment was termed Fragment The plasmid pUCA was prepared by ligating the large fragment obtained by digestion of Fragment (A) with SmaI and the DNA fragment obtained by digestion of the vector, pUC18 (Takara Shuzo, Co., Ltd.) with SmaI.

The plasmid pHSGB was prepared by ligating the large fragment obtained by digestion of Fragment with KpnI and the DNA fragment obtained by digestion of the vector, pHSG399 (Takara Shuzo, Co., Ltd.) with HincII and KpnI.

The plasmid pUCA was digested with KpnI, the blunt-end fragment was prepared with the large fragment 15 of DNA polymerase I (Klenow fragment), and digested with PstI, and finally, a DNA fragment containing Fragment was isolated. Plasmid pHSGB was digested with HindIII, the blunt-end fragment was prepared with the large fragment of DNA polymerase I (Klenow fragment), and digested with PstI, and finally, a DNA fragment containing Fragment was isolated. The plasmid pHSGSK was prepared by ligating both DNA fragments.

The SmaI-KpnI fragment derived from Fragments (A) and in pHSGSK was termed Fragment Fragment (C) corresponded to a fragment obtained by digestion of a 4.8-kb HindIII-HindIII fragment with SmaI and KpnI, contained a promoter, the SD sequence and a upstream region of the ilvG gene, but lost the DNA sequence of 0.2 kb from a leader sequence to an attenuator. The scheme of construction of pHSGSK is summarized in Fig. Fragment was obtained by digestion of the plasmid pHSGSK with SmaI and KpnI, the large DNA fragment was obtained by digestion of the plasmid pBRGM7 with SmaI and KpnI, and the both two fragments were ligated. The obtained plasmid was termed pdGM1. pdGMl harbored a 4.6-kb HindIII-HindIII fragment including the ilvGM gene, which lost the region necessary for attenuation. This ilvGM gene which loses the region necessary for attenuation represents attGM". The scheme of the construction of pdGMl is summarized in Figure 6.

15 The plasmid pDRIA4 described in Japanese Patent Application Laid-Open No. 2-458(1990) is prepared by combining the shuttle vector pDR1120, which allows autonomous replication in both a microorganism belonging to the genus Escherichia and a microorganism belonging to the genus Brevibacterium, with a BamHI-BamHI fragment including the ilvA gene encoding threonine deaminase and a portion of the 3'-terminal of the ilvD gene derived f* from E. coli K-12. Japanese Patent Application Laid- Open No. 2-458(1990) describes that the length of the BamHI-BamHI fragment is 2.3 kb; however, at present, the length of this fragment has been found to be 2.75 kb.

The plasmid pDRIA4 is not present within the chromosomal DNA of Brevibacterium flavum AJ12358 (FERM P-9764) or Brevibacterium flavum AJ12359 (FERM P-9765). From these strains, the plasmid pDRIA4 can be prepared according to the usual method.

From a 2.75-kb BamHI-BamHI DNA fragment in the plasmid pDRIA4, a HindIII-BamHI fragment including the ilvA gene encoding threonine deaminase, in which the inhibition by L-isoleucine was released, was prepared, and ligated to a DNA fragment obtained by cleaving the vector pMW119 (NIPPON GENE) with HindIII and BamHI. The resulting plasmid was termed pMWA1.

A DNA fragment obtained by.cleaving the plasmid pMWA1 with HindIII and a DNA fragment obtained by cleaving the plasmid pdGMl with HindIII were ligated.

15 According to the analysis of the position of the restriction sites of the ligated plasmids, the plasmid in which the transcriptional orientations of the ilvGM and ilvA genes were the same was selected, and termed pMWGMA2. The pMWGMA2 includes the ilvGM gene in which an attenuator was deleted, a 5'-terminal portion of the ilvE gene, and a 3'-terminal portion of the ilvD gene.

The scheme of the construction of pMWGMA2 is summarized in Figure 7.

The chromosomal DNA of Escherichia coli MI162 was prepared and cleaved with SalI and PstI to prepare the mixture of DNA fragments. On the other hand, a DNA fragment was prepared by cleaving the vector pUC19 (Takara Shuzo, Co., Ltd.) with SalI and PstI. The mixture of DNA fragments was ligated to the DNA fragment obtained by cleaving pUC19, and the DNA mixture was obtained. The DNA mixture was induced into AB2070, a transaminase B-deficient strain, (provided from Escherichia coli Genetics Stock Center. J. Bacteriol., 109, 703, (1972), CGSC2070) and a transformant, in which the branched-chain amino-acid requirement was recovered, was selected. As a result of the preparation of a plasmid from the strain, the plasmid harbored a DNA fragment obtained by cleaving the plasmid pUC19 with SalI and PstI, and a.SalI-PstI DNA fragment including the ilvE gene, which were ligated. The plasmid was termed pUCE1. The pUCEl includes a 3'-terminal portion :i 15 of the ilvM gene, the ilvE gene, and a portion of the ilvD gene.

A DNA-fragment mixture was prepared by partially digesting pMWGMA2 with HindIII. On the other hand, a 1.7-kb HindIII-HindIII DNA fragment containing a portion of the ilvE gene and a 5'-terminal portion of the ilvD gene was prepared by cleaving pUCEl with HindIII. Using a DNA mixture obtained by ligating both of the DNA fragments, AB1280, a dihydroxy-acid dehydratase(ilvD gene product)-deficient strain, was transformed, and the strain which recovered branched chain amino acid requirement was selected from the transformants. In the plasmid prepared from the resulting transformant, a DNA 24 fragment obtained by cleaving only the HindIII site between attGM and ilvA of pMWGMA2 with HindIII, and a 1.7-kb HindIII-HindIII DNA fragment including a portion of the ilvE gene and a portion of the ilvD gene derived from pUCE1 were ligated, and the ilvGMEDA operon was reconstructed. The plasmid was termed pMWD5. The scheme of the construction of pMWD5 is summarized in Figure 8.

The resulting plasmid pMWD5 derived from the vector pMW119 harbors the ilvGMEDA operon in which the region necessary for attenuation is deleted.

The plasmid pMWD5 (Apr) obtained as described above is a plasmid containing pMW119 as a vector and carrying the ilvGMEDA operon from which the region 15 required for attenuation was removed.

Production of plasmid containing ilvGMEDA operon and aspA gene pMW118::aspA was digested with SacI and HindIII, and blunt-ended to obtain a DNA fragment containing the o aspA. pMWD5 was digested with AflII, blunt-ended and inserted at the cleavage site with the above DNA fragment containing aspA to obtain pMWD5-aspA (Fig. 9).

Production of plasmid containing ilvGMEDA operon and pntAB gene pMW::THY was digested with SmaI and HindIII, and blunt-ended to obtain a DNA fragment containing pntAB.

was digested with AflII, blunt-ended, and inserted at the cleavage site with the above DNA fragment containing the pntAB to obtain pMWD5-THY (Fig. 9).

Production of plasmid containing ilvGMEDA operon and ppc gene pppc was digested with SacI and XbaI, and bluntended to obtain a DNA fragment containing ppc. was digested with AflII, blunt-ended and inserted at the cleavage site with the above DNA fragment containing ppc to obtain pMWD5-ppc (Fig. 9).

Production of plasmid containing ilvGMEDA operon, 15 pntAB gene and ppc gene pPTS was digested with SacI and HindIII, and blunt-ended to obtain a DNA fragment containing ppc and pntAB. pMWD5 was digested with AflII, blunt-ended, and inserted at the cleavage site with the above DNA fragment containing ppc and pntAB to obtain (Fig. 9).

Production of plasmid containing ilvGMEDA operon, aspA gene, pntAB gene and ppc gene pAPT was digested with SacI and HindIII, and blunt-ended to obtain a DNA fragment containing ppc, pntAB and aspA. pMWD5 was digested with AflII, blunt- 26 ended and inserted at the cleavage site with the above DNA fragment containing ppc, pntAB, and aspA to obtain (Fig. 9).

Example 2: Production of amino acids by Escherichia coli harboring various plasmids Production of L-threonine The various plasmids obtained in Example 1 were each introduced into Escherichia coli VKPM B-3996.

These strains were cultured under the following conditions.

The culture was performed for 38 hours at 37 0

C

with stirring at 114-116 rpm by using a medium having the composition shown in Table 1. Component A, 15 Component B and Component C mentioned in Table 1 were prepared and sterilized separately, and then they were cooled and mixed in a ratio of 16/20 volume of Component A, 4/20 volume of Component B and 30 g/L of Component C.

The results of measurement of the accumulated amounts of L-threonine in the medium are shown in Table 2. It was found that, in L-threonine producing bacteria belonging to the genus Escherichia, L-threonine productivity could be improved by enhancing intracellular THY activity and PEPC activity. Further, it was also found that Lthreonine productivity could be further improved by enhancing AspA activity.

27 Table 1: Threonine production medium A (g/L)

(NH

4 )2SO 4 16

KH

2

PO

4 1 MgSO 4 7H 2 O 1 FeSO 4 7H 2 0 0.01 MnSO 4 4H 2 0 0.01 Yeast Extract (Difco) 2 L-Methionine adjusted to pH 7.0 with KOH and autoclaved at 115 0 C for 10 minute (16/20 volume) B 20% glucose autoclaved at 115 0 C for 10 minute (4/20 volume) C CaCO 3 according to Japanese Pharmacopoeia, subjected to dry sterilization at 180 0 C for 2 days (30 g/L) antibiotics (100 g/L of streptomycin and Rg/L of ampicillin) Table 2 Host Plasmid Accumulated amount of L-threonine (g/L) B-3996 pMW118 14.0 pppc 14.5 pMW::THY 15.0 PMW118::aspA 14.0 pPTS 16.8 pAPT 17.2 0 Production of L-isoleucine The various plasmids obtained in Example 1 were each introduced into Escherichia coli VKPM B-3996.

These strains were cultured under the following conditions.

The culture was performed in a medium for Lisoleucine production (containing 40 g glucose, 16 g of ammonium sulfate, 1 g of monopotassium phosphate, 1 g of V 0z 28 magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate, 0.01 g of manganese chloride tetrahydrate, 2 g of yeast extract and 40 g of calcium carbonate in 1 L of water, pH 7.0) at 37 0 C for 24 hours. L-Isoleucine contained in the medium was quantified by high performance liquid chromatography.

The results are shown in Table 3.

It was found that, in L-threonine producing bacteria belonging to the genus Escherichia, Lisoleucine productivity could be improved by enhancing intracellular THY activity and PEPC activity. Further, it was also found that L-isoleucine productivity could be further improved by enhancing AspA activity.

15 Table 3 Accumulated amount of L- Host Plasmid isoleucine (g/L) B-3996 pMWD5 10.0 pMWD5-ppc 9.9 10.4 10.0 10.8 pMWD5-APT 11.2

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For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

*ooo Sequence Listing esr

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<110> Ajinomoto Co., Inc.

<120> Method for Producing Threonine and Isoleucine <130> OP1188 <140> <141> 2001-08- <150> JP 2000-244921 <151> 2000-08-11 <160> 6 <170> PatentIn Ver. <210> 1 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 1 tgatcagcga aacactttta <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 2 cagcaaacta tgatgagaa <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 3 taacatcact gagatcatgt tg 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 4 tcttttcttg catcttgttc g 21 <210> <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> tctgtttctc aagattcagg ac 22 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 6 cgccggtaaa ccaaaaccc 19 b**.9O 0 0*0* 00** *0 0 0 000 0 0 000*00 0 0 0000 00 000* 0 *000 *00000 00 00 0 0 0

Claims (8)

1. A bacterium belonging to the genus Escherichia, which has an ability to produce L-threonine or L-isoleucine, and in which intracellular phosphoenolpyruvate carboxylase activity and transhydrogenase activity are enhanced.

2. The bacterium belonging to the genus Escherichia according to claim 1, in which activity of an enzyme or enzymes encoded by threonine operon or a part thereof is enhanced, and which has L-threonine producing ability.

3. The bacterium belonging to the genus Escherichia according to claim 2, wherein the threonine operon consists of thrABC. 15

4. The bacterium belonging to the genus Escherichia according to claim 1, in which activity of S.an enzyme or enzymes encoded by ilv operon or a part thereof is enhanced, and which has L-isoleucine producing ability.

5. The bacterium belonging to the genus Escherichia according to any one of Claims 1-4, wherein aspartase activity is enhanced.

6. The bacterium belonging to the genus 5 o Escherichia according to any one of claims 1-5, wherein activity of each enzyme is enhanced by increasing copy number of a gene or operon coding for each enzyme, or modifying an expression regulatory sequence so that intracellular expression of the gene or operon should be enhanced.

7. The bacterium belonging to the genus Escherichia according to claim 6, wherein the gene is derived from a bacterium belonging to the genus Escherichia.

8. A method for producing L-threonine or L- isoleucine, which comprises culturing a bacterium belonging to the genus Escherichia according to any one of claims 1-7 in a medium to produce and accumulate L- threonine or L-isoleucine in the medium, and collecting the L-threonine or L-isoleucine from the medium. Dated this 31st day of July 2001 AJINOMOTO CO., INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *se *@0 0 o